Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine

Definitions

• the present invention relates to methods and compositions useful for inhibiting, treating, protecting, or preventing infection by Actinobacillus suis.
• Actinobacillus suis has recently emerged as a new threat to the swine industry in the United States. Previously associated with high mortality in "high health" herds, A. suis is now recognized as an important pathogen of conventional herds. It is particularly detrimental to younger animals. Infection results in actinobacillosis and can cause sudden death in both neonate and weaned pigs. Symptoms in weaned pigs include anorexia, fever, cyanosis, congestion of the extremities, respiratory distress, pneumonia, necrotizing pneumonia, persistent cough, skin lesions, and fatal septicemia. Pneumonia, arthritis, septicemic signs, pleurisy, pericarditis, and miliary abscesses are known to occur in finishing pigs. Actinobacillosis also causes metritis and abortion in sows.
• the gross pathology of actinobacillosis is characterized by lesions found in lungs, kidney, heart, liver, spleen, intestines and skin; hemorrhages and necrosis; and pneumonic lesions resembling pleuropneumonia. Histopathologically, the disease is characterized by the presence of bacterial thromboemboli with accompanying fibrinohemorrhagic necrosis in the vessels of various tissues; necrotizing bronchopneumonia; and pleuritis.
• Actinobacillus suis is an opportunistic, gram-negative, non-motile, aerobic and facultative anaerobic coccobacillus that colonizes the upper respiratory tract. Genotyping of A. suis isolates recovered from clinical cases in the North American swine herds has revealed a limited genetic variability, with only 13 strains being identified among 74 isolates recovered from 29 different herds. The Simpson's diversity index (also known as species diversity index, see Simpson, 1949) for A.
• A. suis The phenotypic diversity of A. suis is also relatively limited. Only 2 serovars, namely 01 and 02 (Rullo, Papp-Szabo and Michael, 2006), and three capsular types, Kl-3, have been described so far. Pathogenicity studies suggest that isolates from serogroup 02 tend to be more virulent than 01 isolates (Slavic, DeLay and Hayes, 2000). Serotyping of A. suis isolates used for autogenous vaccine production also confirms that a higher percentage of 02 isolates were associated with clinical disease compared with 01 isolates (Slavic, Toffner, and Monteiro, 2000). Although some of the A. suis virulence factors are known (e.g. the RTX toxins Apx I var.
• LPS lipopolysaccharide
• CPS capsular polysaccharides
• OmpA outer membrane protein A
• proteases and iron acquisition.
• compositions of the invention comprise a supernatant collected from one or more A. suis cultures grown to between 0.650 OD 6 5o and 0.850 OD 6 5o; and an adjuvant.
• the supernatant is inactivated, most preferably by formalin.
• the supernatant is also preferably filtered, such as through a 45 micron filter. Filtration may occur before or after inactivation as deemed appropriate for a given situation.
• the supernatant, more preferably the filtered supernatant is essentially free from A suis cells but may contain multiple cellular components.
• adjuvants may be suitably included in a composition of the invention.
• One exemplary adjuvant is Emulsigen® -D.
• the determination of adjuvant will, in part, depend upon the nature of the subject that is to receive the composition; the method of administration to the subject; and conditions under which the composition is to be administered.
• the adjuvant and supernatant, or filtered supernatant may be admixed together prior to administration to a subject, administered simultaneously, or administered sequentially to a subject.
• Suitable subjects of the immunogenic compositions of the invention include animals and humans.
• Animals in which the immune response is stimulated by use of compositions or methods of the invention include livestock, such as pigs, calves, chickens, goats, and sheep, and domestic animals, such as mice, rabbits, dogs, cats, and horses.
• Preferred animals include porcines, murids, equids, lagomorphs, and bovids. Most preferably the animal is a porcine.
• the invention also provides methods of provoking an immune response against Actinobacillus reducing the incidence of or severity of a clinical sign associated with Actinobacillus suis infection in a subject comprising administering to the subject an immunogenic composition of the invention.
• Clinical signs associated with A. suis that may be reduced in incidence or severity in a subject include meningitis, septicemia, metritis, pneumonia, crysipelas-like lesions, and abortion. Any one of which may be lessened by the administration of a composition of the invention relative to a subject not receiving the immunogenic composition.
• Preferred compositions of the invention elicit a protective immunological response that is at least a 10% reduction in at least one clinical sign of an A. suis infection.
• a preferred Actinobacillus suis infection that may be reduced by administration of a composition of the invention is A. suis ISU-8594.
• the invention also provides methods of making or preparing immunogenic compositions of the invention that may be useful in the making of a medicament. Such methods include the steps of growing an Actinobacillus suis culture to between 0.650 ⁇ 6 5 ⁇ and 0.850 OD 65 o; collecting a supernatant from the culture; filtering the supernatant to yield a filtrate; and mixing the filtrate with an adjuvant. Such methods may also include inactivating the supernatant, preferably prior to admixing the supernatant with an adjuvant. Inactivation may occur either before or after filtering the supernatant.
• the invention further provides methods of diagnosing an Actinobacillus suis infection in a subject.
• Such methods comprise: a) providing a filtered supernatant prepared by growing an A. suis culture to between 0.650 OD 6 5o and 0.850 OD 6 5o, collecting supernatant from the culture, and filtering the supernatant; b) contacting the filtered supernatant with a sample obtained from the subject; and c) identifying the subject as having an A. suis infection if an antibody capable of binding a component in the filtered supernatant is detected in the sample.
• binding may be detected by using a second antibody capable of binding the antibody in the sample. Binding by such second antibodies may by detected by a colorimetric assay or other suitable means.
• kits that comprise: a) a filtered supernatant prepared by growing an Actinobacillus suis culture to between 0.650 ⁇ 6 5 ⁇ and 0.850 ⁇ 6 5 ⁇ , collecting a supernatant of the culture, and filtering the supernatant; b) an adjuvant; and c) a container for packaging the supernatant and adjuvant.
• the filtered superantant and adjuvant may be packaged together or separately.
• a kit may further comprise instructions for use of the kit. It may also comprise a means of administering the filtered supernatant and adjuvant to a subject. A means of admixing the supernatant and adjuvant together may also be included in a kit.
• compositions of the invention may further comprise a veterinarily acceptable carrier, second adjuvant, or combination thereof.
• Such compositions may be used as a vaccine and comprise an attenuated vaccine, an inactivated vaccine, or combinations thereof.
• Such vaccines elicit a protective immunological response against at least one disease associated with Actinobacillus .
• Preferred inactivation agents for use in methods of the invention are selected from the group consisting of binary ethyleneimine (BEI) and formalin. Formalin is a more preferred inactivation agent.
• BEI binary ethyleneimine
• Formalin is a more preferred inactivation agent.
• Methods of the invention may also comprise admixing a composition of the invention with a veterinarily acceptable carrier, adjuvant, or combination thereof.
• a veterinarily acceptable carrier for example, a benzyl alcohol, benzyl ether, benzyl ether, benzyl ether, benzyl ether, benzyl ether, benzyl ether, benzyl ether, benzyl ether, sulfate, sulfate, sulfate, or combination thereof.
• compositions of the invention may also be administered in two or more doses, as well as, by other routes of administration.
• routes of administration include subcutaneously, intracutaneously, intravenously, intravascularly, intraarterially, intraperitnoeally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, or intravaginally.
• the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
• kits for vaccinating a subject comprising a set of printed instructions; a dispenser capable of administering a vaccine to an animal; and a supernatant from an A. suis culture having one or more components that effectively stimulates an immune response in a subject.
• Kits of the invention may further comprise a veterinarily acceptable carrier, adjuvant, or combination thereof.
• a dispenser in a kit of the invention is capable of dispensing its contents as droplets; and the A. suis supernatant included in the kit is capable of reducing the severity of at least one clinical sign of an A. suis infection when administered to a subject.
• the severity of a clinical sign is reduced by at least 10% as compared to an untreated, infected subject.
• an "immunogenic or immunological composition” refers to a composition of matter that comprises at least one A. suis supernatant, or immunogenic portion thereof, that elicits an immunological response of a cellular or antibody-mediated immune response to the composition in the subject.
• an immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more of the clinical signs of a Actinobacillus infection.
• an "immune response” or “immunological response” means, but is not limited to, the development of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest.
• an immune or immunological response includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
• the vaccinated subject will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced.
• Such protection will be demonstrated by either a reduction in number of symptoms, severity of symptoms, or the lack of one or more of the symptoms associated with the infection of the pathogen, a delay in the of onset of clinical symptoms, reduced pathogen persistence, a reduction in the overall pathogen load and/or a reduction of pathogen excretion.
• Protection against A. suis means an immune response against A. suis generated by an immunization schedule that results in fewer deleterious effects than would be expected in a non-immunized subject that has not been previously exposed to A. suis. That is, the severity of the deleterious effects of the infection are lessened in an immunized subject because the subject's immune system is resistant to the bacterium. Infection may be reduced, slowed, or possibly fully prevented, in an immunized subject, preferably a pig.
• complete prevention of infection is meant, it is specifically stated. If complete prevention is not stated then the term includes partial prevention.
• “reduction of the incidence and/or severity of clinical signs” or “reduction of clinical symptoms” means, but is not limited to, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects exhibiting clinical signs of infection, or reducing the severity of any clinical signs that are present in the subjects, in comparison to wild- type infection.
• it should refer to any reduction of pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction of any clinical sign symptomatic of A. suis infection.
• these clinical signs are reduced in subjects receiving the composition of the present invention by at least 10% in comparison to subjects not receiving the composition and may become infected. More preferably clinical signs are reduced in subjects receiving the composition of the present invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.
• the term "increased protection” herein means, but is not limited to, a statistically significant reduction of one or more clinical symptoms which are associated with A. suis infection in a vaccinated group of subjects vs. a non-vaccinated control group of subjects.
• the term "statistically significant reduction of clinical symptoms” means, but is not limited to, the frequency in the incidence of at least one clinical symptom in the vaccinated group of subjects is at least 20%, preferably 30% , more preferably 50%, and even more preferably 70% lower than in the non-vaccinated control group after the challenge with an infectious Actinobacillus bacterium.
• compositions used herein may incorporate known injectable, physiologically acceptable sterile solutions.
• aqueous isotonic solutions e.g. saline or plasma protein solutions
• the immunogenic and vaccine compositions of the present invention can include veterinary-acceptable carriers, diluents, isotonic agents, stabilizers, or adjuvants.
• a veterinary-acceptable carrier includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
• stabilizing agents for use in the present invention include stabilizers for lyophilization or freeze-drying.
• the immunogenic composition of the present invention contains an adjuvant.
• adjuvants can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-OlOO (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
• the emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters.
• light liquid paraffin oil European Pharmacopea type
• isoprenoid oil such as squalane or squalene
• oil resulting from the oligomerization of alkenes in particular of isobutene or decene
• the oil is used in combination with emulsifiers to form the emulsion.
• the emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121.
• mannide e.g. anhydromannitol oleate
• glycol of polyglycerol
• propylene glycol and of oleic isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products
• an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.
• Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Patent No.
• 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms.
• the preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups.
• the unsaturated radicals may themselves contain other substituents, such as methyl.
• the products sold under the name Carbopol ; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol.
• Carbopol 974P, 934P and 97 IP there may be mentioned Carbopol 974P, 934P and 97 IP. Most preferred is the use of Cabopol 97 IP.
• copolymers of maleic anhydride and alkenyl derivative are the copolymers EMA (Monsanto), which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.
• Suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof or stimulants of endogenous cytokine release, among many others.
• Disposents can include water, saline, dextrose, ethanol, glycerol, and the like.
• Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
• Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.
• an adjuvant can be added in an amount of about 100 ⁇ g to about 10 mg per dose, preferably in an amount of about 100 ⁇ g to about 10 mg per dose, more preferably in an amount of about 500 ⁇ g to about 5 mg per dose, even more preferably in an amount of about 750 ⁇ g to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose.
• the adjuvant may be at a concentration of about 0.01 to 50%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to 25%, still more preferably at a concentration of about 7% to 22%, and most preferably at a concentration of 10% to 20% by volume of the final product.
• an effective dose means, but is not limited to, an amount of A. suis supernatant or filtered supernatant that elicits, or is able to elicit, an immune response that yields a reduction of clinical symptoms in a subject to which the supernatent is administered.
• Safety refers to the absence of adverse consequences in a vaccinated subject following vaccination, including but not limited to: potential reversion of a bacterium-based vaccine to virulence, clinically significant side effects such as persistent, systemic illness or unacceptable inflammation at the site of vaccine administration.
• vaccination or “vaccinating” or variants thereof, as used herein means, but is not limited to, a process which includes the administration of a composition of the invention that, when administered to a subject, elicits, or is able to elicit— directly or indirectly— an immune response in the subject against A. suis.
• Methods for the treatment or prophylaxis of infections caused by A. suis are also disclosed.
• the method comprises administering an effective amount of the immunogenic composition of the present invention to a subject, wherein said treatment or prophylaxis is selected from the group consisting of reducing signs of A. suis infection, reducing the severity of or incidence of clinical signs of A. suis infection, reducing the mortality of subjects from A. suis infection, and combinations thereof.
• Mortality in the context of the present invention, refers to death caused by A. suis infection, and includes the situation where the infection is so severe that an animal is euthanized to prevent suffering and provide a humane ending to their life.
• Attenuation means reducing the virulence of a pathogen.
• attenuation is synonymous with "avirulent".
• an attenuated bacterium is one in which the virulence has been reduced so that it does not cause clinical signs of an A. suis infection but is capable of inducing an immune response in the target subject, but may also mean that the clinical signs are reduced in incidence or severity in subjects infected with the attenuated A. suis in comparison with a "control group" of subjects infected with non-attenuated A. suis and not receiving the attenuated bacterium.
• the term "reduce/reduced” means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, still more preferably 80%, even more preferably 90% and most preferably of 100% as compared to the control group as defined above.
• an "effective amount" for purposes of the present invention means an amount of an immunogenic composition capable of inducing an immune response that reduces the incidence of or lessens the severity of A. suis infection in a subject.
• An effective amount refers to colony forming units (CFU) per dose or logs/dose.
• Long-lasting protection shall refer to "improved efficacy” that persists for at least 3 weeks, but more preferably at least 3 months, still more preferably at least 6 months. It is most preferred that the long lasting protection shall persist until the average age at which porcine animals are marketed for meat.
• FIG. 1 Daily average temperatures among treatment groups from day 0 to day 41 of the study. DETAILED DESCRIPTION
• the invention provides methods and compositions useful for inhibiting, treating, protecting, or preventing infection by Actinobacillus suis.
• A. suis prototype vaccines against a heterologous challenge of various serotypes or isolates of A. suis. These vaccines were demonstrated to usefully stimulate immunogenic reactions in treated animals. Some vaccines stimulated reactions sufficient to be protective against A. suis.
• the A. suis vaccine prototypes were comprised of different, culture fractions (e.g. whole cell, supernatant, or outer membrane protein (omp)) and a Ingelvac® APP-ALC live vaccine against A. pleuropneumoniae (APP).
• the whole cell vaccine was made from the pelleted material that resulted from a centrifugal spin of a cell culture.
• the cultural supernatant was made from the supernatant that resulted from the centrifugal spin, and may have included exotoxins, endotoxins, secreted or sloughed-off omps, and other cellular components that were not removed during the spin (see Example 1).
• the Ingelvac® APP-ALC live vaccine was included because APP encodes Apx I and II toxins that are very similar to A. suis toxins. It was hypothesized that the Ingelvac® APP-ALC vaccine may provide some cross protection against A. suis.
• Treatment group 1 (15 pigs) received a 1 x 2mL intramuscular (IM) dose of a formalin-inactivated, whole cell fraction of A. suis on days 0 and 21 of the study, respectively.
• Treatment group 2 (15 pigs) received a 1 x 2mL IM dose of a formalin-inactivated A. suis culture supernatant fraction on days 0 and 21 of the study, respectively.
• Treatment group 3 (15 pigs) received a 1 x 2mL IM dose of an A. suis outer membrane protein (omp) cellular fraction on days 0 and 21 respectively.
• IM intramuscular
• Treatment group 4 (15 pigs) received a 1 x 2mL IM dose of Ingelvac® APP-ALC on days 0 and 21 respectively.
• Treatment group 5 (15 pigs) designated as “challenge controls” received a 1 x 2mL IM dose of placebo on days 0 and 21 respectively.
• Treatment group 6 (10 pigs) were designated “strict controls” and did not receive vaccine or placebo treatment.
• Fresh and fixed (lungs only) samples of the lung (3 lobes with lesions, if present), liver, kidney, spleen, tonsil, and swabs of nasal turbinates, trachea, bronchi, meninges, and heart blood were collected from each animal and were cultured for bacterial isolation and histopathology (lungs only).
• the A. suis fractions and placebo groups were adjuvanted with Emulsigen® -D, and a few adverse injection site reactions were noted in these groups.
• One pig in the supernatant group (group 2) was noted with a mild reaction at the injection site during the first vaccination event.
• the Emulsigen® -D in combination with the test articles did not produce any significant injection site reactions in the supernatant and omp groups.
• the whole cell group did have a few animals with abscesses present at the time of necropsy, which could have been due to the test article formulation that consisted of a concentrated whole cell stock.
• the administration of these vaccine prototypes i.e. whole cell, supernatant, and omp
• the APP-ALC group had some injection site swelling, which is consistent with the product literature that notes swelling in 11% of the vaccinated animals.
• the secondary parameters monitored were used to support the primary efficacy parameters (i.e., gross and microscopic (IHC) lesions) responses due to vaccine and challenge for each treatment group.
• Clinical observations made during the challenge period indicated that the challenge controls had an overall higher score when compared to the vaccine treatment groups 1-4, showing that the challenge controls responded to the challenge.
• the supernatant group did not seem to be as clinically affected. However, all groups did have some pigs that did have some respiratory issues with a cough, body condition, or behavior.
• the treated groups saw an increase in average rectal temperatures, above 104 °F, four hours after the first vaccination event. Temperatures for the APP and omp groups spiked at or above 104.9 °F, which was the cut-off of pyrexia following four hours post vaccination, and lasted about twenty-four hours for the omp group. Temperatures in the groups were returning back to normal by day 1 or 3. For the booster event, temperatures were only taken on day 28 and not at 4hrs or on days 1, 3, and 5 post-vaccination as with the first vaccination. During the booster period, on day 28 the APP group was statistically different (p ⁇ 0.05) when compared to the whole cell and supernatant groups.
• the challenge controls had an overall higher rectal temperature at both the beginning and end of the period.
• the whole cell group had the highest rectal temperatures during days 38 and 39.
• the challenge control group had the highest overall percent animals febrile (10/15) followed by the whole cell (8/15), OMP (5/15), supernatant (2/15), and APP (1/15) groups, respectively.
• EXAMPLE 1 Prototype Vaccines and Challenge Treatment A. Preparation of Vaccine Prototypes
• A. suis (KSU-1) cultures One milliliter of A. suis (103007-1KSU) lot # 201-9,10,11 was used to inoculate 150 ml of BHI media (lot # 195-121) in a 250 ml spinner. The inoculated media was placed at 37°C at about 72 rpm for six hours. Afterwards, about 2.5 ml of the inoculated media was added to about 2500 ml fresh BHI media. This step was repeated until four batches of 2500 ml were prepared. The four batches were incubated at 37°C overnight at about 72 rpm. Two batches were used to isolate whole cell OMP, and supernatant fractions, one batch was used to prepare a concentrated whole cell fraction, and the final batch was used for LPS extraction.
• the filtered supernatant was aliquoted into 2 x 850 cm RBs, 81 x 5 mL vials, and 4 x 250 mL Nalgene bottles. Filtered supernatant was stored at -20°C until use.
• A. suis culture was harvested as described above. The pellets were resuspended in lx PBS buffer and re-centrifuged at 10K rpm for ten minutes. Supernatants were decanted, and the pellets were resuspended in 1 L of lx PBS buffer. An optical density reading at about 600 nm was 1.003, which is within the expected range of 0.8-1.200 for LPS extraction.
• the LPS fraction was aliquoted into 3 x 250 mL Nalgene bottles and stored at -80°C until use. The remaining volume of LPS fraction was stored at 4°C for LPS extraction.
• KSU-1 Whole cell formalin-inactivated vaccine The optical density (OD) of the cell culture measured at 600 nanometer wavelength was at 0.775 at the time of harvest. In previous studies, this optical density has correlated to approximately 8.7 logs/mL based on the logarithmic estimate curve to determine logs/mL based on colony forming units (cfu). The whole cell fraction was concentrated lOx and frozen. The frozen stock was then formalin- inactivated, and diluted (54mLs of culture into 26 mLs of lx phosphate buffered saline (pbs), to 67.5% of the total fraction. The inactivated, diluted vaccine stock was then stored at 4°C.
• the whole cell formalin-inactivated vaccine stock was removed from 4°C for formulation.
• 80mLs of the material was aliquoted out into a sterile lOOmL Pyrex bottle containing a magnetic stir bar.
• the bottle was placed on a stir plate, and with continuous stirring, 20 mLs (20% v/v) of the adjuvant (Emulsigen® -D) was added over 1 minute.
• the adjuvanted prototype was then stirred for an additional 10 minutes.
• the material was transferred to a 100 mL vaccine bottle where it was capped and placed at 4°C. Lot nos. for the whole cell formalin-inactivated vaccine prototype were N201-110-WC-073108 (day 7) and N201-122-WC-082108 (day 21).
• Sterility was verified by no growth on 5% Sheep Blood Agar Plate (BAP) after 28hrs and 30hrs of 37°C aerobic incubation for day 0 and day 21 respectively.
• Vaccine prototype material was kept on ice prior to administration to the test animals and during the entire vaccine administration procedure.
• KSU-1 Supernatant formalin-inactivated vaccine The supernatant prototype was formulated from the lOx concentration step of the whole cell fraction preparation (see above) by collecting the supernatant and filtering it through a 0.45 micro filter. The resulting filtered supernatant was frozen. This supernatant was then formalin inactived and stored at 4°C until formulated. The formulation step was as for the whole cell formalin-inactivated vaccine. Lot nos. for the supernatant formalin-inactivated vaccine prototype were N201-110-Supe-073108 (day 7) and N201-122-Supe-082108 (day 21).
• KSU-1 OMP fraction vaccine The OMP fraction was produced from pellets prepared from a KSU-1 culture (isolate BI- 103007- IKSU) and stored at -80°C by using the following OMP sarcosyl extraction procedure. Pellets were thawed on the benchtop. The pellets were resuspended (22.5g wet weight) with 130 mL of 10 mM HEPES balanced salt solution and aliquoted into four 50 mL conical Falcon tubes (40 mL/tube).
• the pooled supernatants were run in an ultra centrifuge, -0.0128 kg/tube, at a setting of 124,000 x g (30,000 rpm), 1: 10 hours, 4°C, with maximum acceleration, and no brake. Supernatants were decanted, pooled with the earlier supernatant, and stored at 4°C. About 1 mL of lOmM HEPES was added on top of each pellet in the ultra centrifuge tubes, and the pellets were incubated overnight at 4°C to loosen the pellets from the tubes. Another 2 mL of lOmM HEPES was added to each tube to resuspend the pellets. Each resuspended pellet solution was q.s.
• Duplicate samples (1: 10 dilution) were run on either a reduced MOPS 10-12% Bis- Tris SDS PAGE gel or a NuPAGE 4-12% Bis-Tris gel to visualize protein bands.
• Total protein concentrations of the final OMP stock were determined using the BCA and were determined prior to formulation with adjuvant. Based on the values obtained by BCA, the total mass of protein contained in the OMP extraction stock was 1.75 ⁇ g/mL. Based on this value, the potency of the formulated vaccine OMP prototype was 250 ⁇ g/dose.
• the resuspended, extracted OMP fraction was aliquoted into 200 ⁇ samples, labelled, and frozen at -70°C.
• the OMP prototype vaccine was transferred to a 60 mL vaccine bottle where it was capped and placed at 4°C. Lot nos. for the OMP vaccine prototype were N201-110-OMP- 073108 (day 7) and N201-122-OMP-082108 (day 21).
• Ingelvac® APP-ALC vaccine One bottle of APP-ALC was removed from -70°C and thawed in luke warm water. Once thawed, a sample was removed using a 16 gauge sterile needle and placed in a vaccine bottle for potency testing. The vaccine bottle was placed at 4°C. Titrations of Ingelvac APP-ALC were performed on Days 0 and 21 for determining colony forming units (CFU) per dose. The samples were serially diluted out 10-fold and plated onto Mueller Hinton Chocolate Agar plates. Three reps were performed and allowed to incubate at 37°C for 24hrs before determining the CFU.
• CFU colony forming units
• Colony forming units were respectively at Day 0: 9.99 logs/dose and at Day 21: 10.2 logs/dose.
• Lot nos. for the Ingelvac® APP-ALC vaccine were N201-110-APP-073108 (day 0) and N201-122-APP-082108 (day 21).
• Vaccine prototype material was maintained prior to administration to the test animals and during the entire vaccine administration procedure as previously described.
• Placebo vaccine Working in a bio-safety hood, 60 mLs of lx phosphate buffered saline (pbs) was aliquoted out into a sterile 100 mL Pyrex bottle containing a magnetic stir bar. The bottle was placed on a stir plate, and with continuous stirring, 15 mLs (20% v/v) of the adjuvant (Emulsigen® -D) was added over 1 minute. The adjuvanted prototype was then stirred for an additional 10 minutes. The material was transferred to 100 mL vaccine bottle where it was capped and placed at 4°C. The Lot no. for the placebo vaccine was N 201-109. [0085] Sterility was verified, and the placebo prototype material was maintained prior to administration to the test animals and during the entire vaccine administration procedure as previously described.
• pbs lx phosphate buffered saline
• the challenge strain used was A. suis Isolate ISU-8594 p5.
• the challenge material was produced by inoculating a 1 liter Belco spinner flask containing 800mLs of Brain Heart Infusion Porcine (BHI-Porcine) media with 5mLs of ISU-8594 p4.
• BHI-Porcine Brain Heart Infusion Porcine
• the spinner was placed into a 37° C incubator at -100 rpm.
• the culture was grown to an optical density (OD600) of 0.109— about 5 hrs and 30 minutes post inoculation.
• the culture was then transferred to 6 x 100 mL vaccine bottles containing 100 mLs total volume that were stoppered and capped.
• the titer of the challenge material was determined by CFU count generated from plating serial 10-fold dilutions onto 5% sheep blood agar plates.
• the challenge titer obtained was 1.69 x 10 cfu(s)/mL or 9.0 logs/6 mL dose.
• the Lot no. for the challenge material was N201-138.
• Challenge material was kept on ice prior to administration to the test animals and during the entire challenge procedure.
• a mixture of female and neutered male porcine animals of a commercial cross and 3 weeks +7 days of age were obtained from Wilson Prairie View Farms. Animals were healthy with no evidence of clinical respiratory disease and negative for bacterial respiratory pathogens such as A. pleuropneumonia, H. parasuis, S. suis, P. multocida, B. bronchiseptica, E. rhusiopathiae and A suis. While allowance was made for animals to be excluded from the study if and when health problems unrelated to vaccination or challenge became apparent, no animals were removed.
• a computer random number generator (Microsoft Office Excel) was used to assign each animal a unique random number. The random number was then sorted into ascending order. Assignments occurred by allocating the experimental units across treatment groups starting with the lowest block of random numbers and assigning them to treatment groups 1 through 6. Increasing in random number, the next block of experimental units were then assigned to treatment groups and so on until all animals were assigned to a treatment group.
• Treatment group 1 (15 pigs) received a 1 x 2mL intramuscular (IM) dose of formalin-inactivated whole cell A. suis on days 0 and 21 of the study, respectively.
• Treatment group 2 (15 pigs) received a 1 x 2 mL IM dose of formalin-inactivated A. suis culture supernatant on days 0 and 21 of the study, respectively.
• Treatment group 3 (15 pigs) received a 1 x 2mL IM dose of A. suis outer membrane protein (omp) on days 0 and 21 respectively.
• Treatment group 4 (15 pigs) received a 1 x 2mL IM dose of Ingelvac® APP-ALC on days 0 and 21 respectively.
• Treatment group 5 (15 pigs) designated as “challenge controls” received a 1 x 2mL dose of placebo by IM route of administration on days 0 and 21 respectively.
• Treatment group 6 (10 pigs) were designated “strict controls” and did not receive vaccine or placebo treatment. All groups were observed for 41 days.
• Groups 1-5 received a 6mL dose/pig (3mL applied to each nostril per pig) of A. suis strain ISU-8594 via intranasal (IN) inoculation containing 1 x 10 9 0 logs/dose.
• Rectal temperatures were collected from all animals prior to treatment on Day 0; 4 hours post inoculation; and on 1 day(s) post inoculation (DPI), 3, 5, 7, 14, 21, 28, 34, 35, 36, 37, 38, 39, 40 and 41 DPI.
• DPI 1 day(s) post inoculation
• Venous whole blood (6-10 mL) was collected from each animal prior to treatment on Day 0 and weekly (Days 7, 14, 21, 28, 34 and 41) for future serological testing.
• Clinical observations were performed daily from Day 0 to Day 41 for any signs of respiratory distress including labored breathing, sneezing, coughing, altered respiratory movements, anorexia, lameness, swelling of joints, dehydration, ability to stand, paddling, moribund for 2 or more consecutive days, or death.
• Animal ID #32 (treatment group 1) was found dead on day 36 one day post-challenge. Gross examination showed that this pig's lungs had severe fibrinouses and fluid in the chest cavity. Its lungs had consolidation and large hemorrhagic areas. The presumptive diagnosis was acute death due to A. suis challenge. Bacteriology cultures were performed on the fresh tissue and swab samples. Actinobacillus suis was recovered in all tissue and swab samples except the spleen and trachea. Histopathology on fixed lung samples indicated evidence of severe pneumonia, necrosis, pleuritis, and bacterial colonies present.
• Injection site reactions for IM-vaccinated treatment groups 1, 2, 3 and 4 were as follows: Pigs #35 (supernatant, group 2) and #10 (APP, group 4) had swelling of a 1 x 1 cm area recorded on days 1, 2, and 3 of the study. No injection site evaluations were recorded for the day 21 vaccination event. Clinical observations revealed swelling in the neck in pig #10 (APP, group 4) on days 4 and 5, pig #20 (APP, group 4) on days 26 and 29, pig #40 (APP, group 4) on days 19 through 35, pig #41 (APP, group 4) on days 26 through 35, and pig #46 (APP, group 4) on days 14 through 35. All remaining animals had a normal, healthy disposition during this observation period.
• Table 1 Average gross lung lesion scores by treatment group and number of animals with a positive gross score within groups.
• Treatment group 5 (challenge), received the highest average gross lung lesion score (28.67) and percent with a lung lesion score (86.67%) compared to the other treatment groups.
• Treatment group 5 was significantly different (p ⁇ 0.05) from treatment group 6, when comparing average gross lung lesion scores and percent positive with a lesion.
• Treatment group 2 (supernatant) received the lowest lesion score (12.68), but had a percent lesion score that was the second lowest (60.00%) compared to treatment group 4 (APP) which had the lowest percent lesion score (50.00%).
• treatment group 1 (whole cell) had the second highest average lung lesion score of 26.73 followed by the treatment group 3 (omp) with a score of 21.69.
• treatment group 3 (omp) had the second highest number of percent lesions present with 80.00% followed by treatment group 1 (whole cell) with 66.67%.
• Lung sections were collected, fixed, and submitted to the Iowa State Veterinary Diagnostic Laboratory for evaluation of non-specific microscopic lesion development (i.e. pneumonia, necrosis, and pleuritis) and presence of bacterial colonies. Scores were based on a nominal scale of 1 to 3 with 1 being mild, 2 being moderate, and 3 being severe. Groups were compared based on average lesion severity and presence of bacterial colonies (bugs) present within each tissue, and the frequency of positives for each group are shown in Table 2. Table 2. Average microscopic lesion scores by treatment group and number of animals with a positive microscopic score within groups.
• Treatment group 2 (supernatant) had the same lowest scores in bronchopneumonia and pleuritis parameters as group 4, and group 2 and group 1 (whole cell) had the same frequency of bacterial colonies present. No statistical differences were found for percent positives in the parameters listed in Table 2.
• treatment group 5 (challenge) had the highest overall score of 1.148 followed by groups 1, 3, 4, and 2 with scores of 1.068, 1.058, 1.036, and 1.017 respectively. For overall average clinical scores, statistical differences (p ⁇ 0.05) were found when comparing groups 1-4 vs. group 5.
• treatment group 5 (challenge) had the highest scores on days 37 through 41.
• Treatment group 1 (whole cell) had the highest score on day 36.
• treatment group 1 (whole cell) scores fell numerically following day 36 spike from 1.25 to a range of 1.00 to 1.07.
• treatment group 5 had the highest numerical rectal temperatures on days 36, 37, 40, and 41; whereas, treatment group 1 (whole cell) had the highest rectal temperatures on days 38 and 39. Furthermore, treatment group 4 (APP) had the lowest overall rectal temperatures on days 36, 37, 38, and 41. In addition treatment group 5 (challenge) had the same lowest temperature on day 38 as treatment group 4, and groups 2 and 3 had the lowest temperatures on days 39 and 40 respectively. Both groups 3 and 5 had an initial rise in temperature one day post-challenge that then fell for group 3; however, the temperature for group 5 rose again on day 40.
• Treatment group 6 and Spleen, Meninges, and Heart Blood were not part of the statistical analysis.
• Swabs of each tissue were used to inoculate agar plates for bacterial isolation. Plates were incubated along with the swab samples mentioned above, and plates were observed for presence of A. suis 24 hours post incubation. Biochemical analysis was done on a random sample in each of the treatment groups to confirm the presence of A. suis. Statistical analyses were only performed to compare groups 1 through 5 on bacterial isolation scores using the Fisher's Exact Test. The spleen, meninges, and heart blood were not statistically analyzed. Tables 7a and 7b provides a summary of the bacteriology results. Table 7a. Number of percent positive A. suis animals in treatment groups by bacterial isolation.
• Table 7b Number of percent positive A. suis animals in treatment groups by bacterial isolation.
• Bordettella sp. was in high numbers in all groups as well as alpha and beta streptococcal bacteria and Pasteurella multocida.
• Other organisms isolated in very low numbers were Arcanobacterium pyrogenes, E. coli, Staphylococcus sp, Enterococcous sp, Proteus, blue fungii, and Pastunella sp.
• compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the following claims.

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